JB is a CRUK clinical fellow, AS is a Wellcome Trust PhD student, the SR lab is funded by a CRUK Senior Research Fellowship (C99667/A12918). This work was supported by two Wellcome Trust Strategic Awards (097945/B/11/Z and 095931/Z/11/Z).

1.&#160;Cell Culture and Treatment
<ol>
	<li>Gather the reagents and equipment listed in Table 1 to culture human osteosarcoma (U2OS) cells. Prepare all reagents and conduct all tissue culture techniques in the laminar air flow hood to ensure sterility</li>
	<li>Prepare the culture medium as follows: Add 50 ml FCS, 5 ml L-glutamine and 5 ml penicillin/streptomycin to the DMEM to achieve final concentrations of 10% (FCS), 2 mM (L-glutamine), 50 U/ml (penicillin) and 50 U/ml (streptomycin). Warm the culture medium at 37 &#176;C&#160;in a water bath.</li>
	<li>Prepare the cells for treatment:
	<ol>
		<li>Sterilize several coverslips in 70% ethanol, allow to air dry and place one on the bottom of six 3.5 cm plates.</li>
		<li>Immerse the coverslips in 2 ml warmed complete DMEM (37 &#176;C), pressing down with the coverslip forceps to ensure that they remain adhered to the bottom of the wells.</li>
		<li>Detach the U2OS cells from their tissue culture plate by washing once with 3 ml PBS, applying 4 ml warmed 0.05% trypsin-EDTA (1%) and incubating at 37 &#176;C for 7 min.</li>
		<li>Resuspend cells in 6ml warmed complete DMEM to inactivate the trypsin-EDTA and count using a hemocytometer.</li>
		<li>Plate 2 x 105 cells to each of the 3.5 cm plates prepared in point 1.3.1. Gently rock the plate to ensure an even cell distribution. Allow the cells to adhere overnight prior to treatment.</li>
	</ol>
	</li>
	<li>Treat the cells by placing four 3.5 cm plates into the hypoxia workstation and leave the other two 3.5 cm plates in the incubator at 37 &#176;C in normal oxygen conditions. Be ready to commence the RNA labelling assay under treated conditions&#160;(in the hypoxia chamber for hypoxia treated cells)&#160;1 hr&#160;before the hypoxia treatment ends.</li>
	<li>Expose cells to hypoxia for 1 hr 15 min, 1 hr 30 min, 2 hr, and 24 hr. These time points are flexible and user-determined.</li>
	<li>Use a normoxic 3.5 cm plate as the positive control and a normoxic 3.5 cm plate treated with Actinomycin D (10 &#181;g/ml final concentration) for 4 hr&#160;as the negative control. Actinomycin D is an inhibitor of global RNA synthesis.</li>
</ol>
Cautions:
Hoechst 33342 is a known mutagen. 
DMSO is known to facilitate the entry of organic molecules into tissues. 
NaOH is corrosive. 
HCl is corrosive. 
Paraformaldehyde is highly toxic to all animals and can cause death in humans. 
Triton X-100 can cause skin irritation following direct contact. 
Actinomycin D is toxic when in contact with skin or swallowed. 
Take appropriate precautions when handling all hazardous chemicals.
2.&#160;Click-iT Assay
<ol>
	<li>Gather the following reagents that are required in addition to the Click-iT assay kit: phosphate buffered saline (PBS) pH 7.2 - 7.6, 3.7% paraformaldehyde (PFA) in PBS, 1% Triton X-100 in PBS, deionized water (dH2O),&#160;dimethyl sulfoxide (DMSO), 10 M sodium hydroxide (NaOH), and 37% hydrochloric acid (HCl); Optional - pH indicator strips.</li>
	<li>Prepare a fresh PFA stock solution before performing the Click-iT reaction as follows:
	<ol>
		<li>Put 1.85 g PFA, 3.5 ml dH2O and 10 &#181;l of10 M NaOH into a 50 ml Falcon tube. Boil 300 - 400 ml of H2O in a large beaker in the microwave to make a water bath and stand this in a fume hood.</li>
		<li>Place the 50 ml Falcon into the water bath and gently agitate for 10 min, periodically releasing the lid until the PFA has dissolved.</li>
		<li>Syringe the PFA solution through a 0.2 &#181;m filter into another 50 ml Falcon tube, resulting in a 37% solution.</li>
		<li>Dilute this 10x&#160;by adding PBS and bring the pH to 6.8 by adding about 12 &#181;l of concentrated HCl. Option: Confirm the correct pH with the indicator strips in the fume hood.</li>
		<li>Use immediately or store overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare the stock solutions of the RNA imaging kit as follows:
	<ol>
		<li>Add 373 &#181;l dH2O to component A resulting in a stock solution of 100 mM of EU. Store at -20 &#176;C for up to one month.</li>
		<li>Add 85 &#181;l of DMSO to component B (Alexa Fluor 594) and mix by pipetting or vortexing. Store both at -20 &#176;C for up to one year.</li>
		<li>Add 2 ml dH2O to component E to create a 10X solution of the RNA imaging kit reaction buffer additive. Store at -20 &#176;C for up to one year, discard when solution develops a brown color.</li>
	</ol>
	</li>
	<li>Labeling of Cells with EU: Prepare a 2X working solution of EU from the 100 mM stock prepared in step 2.3 in prewarmed complete medium (37 &#176;C). Perform the remainder of this step and step 2.5 in the hypoxia chamber for cells treated with hypoxia.&#160;Dilute to 1X by adding an equal volume of this 2X working solution to the media containing cells. Incubate for 1 hr under treatment cell culture conditions.</li>
	<li>Cell Fixation: Wash each well once with PBS and add 1 ml of the 3.7% PFA stock prepared in step 2.2 above under treatment conditions. Incubate for 15 min&#160;under treatment conditions. The samples treated with hypoxia can be removed from the hypoxia chamber at this point. Remove the fixative and wash each well once with PBS (Take care to dispose of PFA waste responsibly). Perform the next steps at room temperature.</li>
	<li>Permeabilization: Remove the wash solution and add 1 ml of 1% Triton X-100 in PBS to each well. Incubate for 15 min&#160;at room temperature. Remove the permeabilization buffer and wash each well once with PBS.</li>
	<li>Labeled RNA Detection:
	<ol>
		<li>Prepare a fresh 1X working solution of RNA imaging kit reaction buffer additive by diluting the 10X solution (prepared in step 2.3) 1:10 in dH2O.</li>
		<li>Prepare the RNA imaging kit reaction cocktail according to Table 2 for use immediately after preparation.</li>
		<li>Remove the wash solution and add 500 &#181;l of RNA imaging kit reaction cocktail to each sample. Incubate for 30 min&#160;at room temperature, protect from light.</li>
		<li>Remove the RNA imaging kit reaction cocktail and wash once with 1 ml of RNA imaging kit reaction rinse buffer (component F), then remove the RNA imaging kit reaction rinse buffer.</li>
	</ol>
	</li>
	<li>Optional multiplex reaction: Perform additional antibody labeling of samples at this point according to manufacturer&#39;s recommendations.</li>
	<li>DNA Staining: Wash samples with PBS then remove the wash solution. Dilute the Hoechst 33342 (Component G) 1:1,000 in PBS. Add 1 ml of diluted Hoechst 33342 to each well and incubate for 15 min&#160;at room temperature, protect from light. Remove the Hoechst 33342 solution and wash the cells twice with PBS. Remove the wash solution and proceed to light microscopy.</li>
</ol>
3.&#160;Light Microscopy
<ol>
	<li>Mount Coverslips:
	<ol>
		<li>Place 10 &#181;l of mounting medium on the center of a standard microscope slide.</li>
		<li>Place the coverslip with cell-side down on the center of the microscope slide to cover the aliquot of mounting medium. Take care to avoid the generation of bubbles in the mounting medium as this will obscure subsequent image acquisition.</li>
		<li>Stick the coverslip to the microscope slide by applying clear nail varnish to its perimeter. Allow to dry for 5 min&#160;at room temperature. Protect from light. Samples may be stored at -20 &#176;C following mounting.</li>
	</ol>
	</li>
	<li>Image acquisition: Acquire images using a wide-field microscope capable of fluorescence detection. Approximate fluorescence/excitation emission maxima for the Alexa Fluor dye and Hoechst 33342 bound to DNA are shown in Table 3.</li>
	<li>Perform Imaging using a 40X/1.30 NA oil immersion lens and capture images with a cooled CCD camera.</li>
</ol>
4.&#160;Data Analysis
<ol>
	<li>Deconvolve images using image processing software (see Materials table) prior to importing to the OMERO client. Normalize rendering settings for all images in the same experiment in the microscope image visualization and analysis software by applying the rendering settings from control normoxic cells to all other conditions.</li>
	<li>Use the Region Of Interest (ROI) tool in the microscope image visualization and analysis software (see Materials table) to select nuclei from each image. Obtain mean intensities for each ROI and calculate the average and standard deviation for each condition by choosing the intensity analysis option, including a number between 30-50 cells.</li>
	<li>Construct Scatter plots using a data graphing software (see Materials table) by plotting all individual mean intensity values obtained in step 4.2 to reflect variability between cells under each condition. Alternatively construct bar graphs to represent average mean intensity plus standard deviation of nascent RNA.</li>
</ol>

<ol>
	<li>Kenneth, N. S., Rocha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+gene+expression+by+hypoxia.">Regulation of gene expression by hypoxia.</a> Biochem J. 414, 19-29 (2008).</li><li>Appelhoff, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+function+of+the+prolyl+hydroxylases+PHD1,+PHD2,+and+PHD3+in+the+regulation+of+hypoxia-inducible+factor.">Differential function of the prolyl hydroxylases PHD1, PHD2, and PHD3 in the regulation of hypoxia-inducible factor.</a> J Biol Chem. 279, 38458-38465 (2004).</li><li>Lancaster, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+dimerization+and+substrate+phosphorylation+inhibit+factor+inhibiting+hypoxia-inducible+factor+(FIH)+activity.">Disruption of dimerization and substrate phosphorylation inhibit factor inhibiting hypoxia-inducible factor (FIH) activity.</a> Biochem J. 383, 429-437 (2004).</li><li>Rocha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulation+under+low+oxygen:+holding+your+breath+for+transcription.">Gene regulation under low oxygen: holding your breath for transcription.</a> Trends Biochem Sci. 32, 389-397 (2007).</li><li>Schodel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+genome-wide+mapping+of+HIF-binding+sites+by+ChIP-seq.">High-resolution genome-wide mapping of HIF-binding sites by ChIP-seq.</a> Blood. 117, (2011).</li><li>Jao, C. Y., Salic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+RNA+transcription+and+turnover+in+vivo+by+using+click+chemistry.">Exploring RNA transcription and turnover in vivo by using click chemistry.</a> Proc Natl Acad Sci U S A. 105, 15779-15784 (2008).</li><li>Breinbauer, R., Kohn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Azide-alkyne+coupling:+a+powerful+reaction+for+bioconjugate+chemistry.">Azide-alkyne coupling: a powerful reaction for bioconjugate chemistry.</a> Chembiochem. 4, 1147-1149 (2003).</li><li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioconjugation+by+copper(I)-catalyzed+azide-alkyne+[3+++2]+cycloaddition.">Bioconjugation by copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition.</a> J Am Chem Soc. 125, 3192-3193 (2003).</li><li>Rostovtsev, V. V., Green, L. G., Fokin, V. V., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stepwise+huisgen+cycloaddition+process:+copper(I)-catalyzed+regioselective+"ligation"+of+azides+and+terminal+alkynes.">A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes.</a> Angew Chem Int Ed Engl. 41, 2596-2599 (2002).</li><li>Kolb, H. C., Finn, M. G., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+Chemistry:+Diverse+Chemical+Function+from+a+Few+Good+Reactions.">Click Chemistry: Diverse Chemical Function from a Few Good Reactions.</a> Angew Chem Int Ed Engl. 40, 2004-2021 (2001).</li><li>Allan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OMERO:+flexible,+model-driven+data+management+for+experimental+biology.">OMERO: flexible, model-driven data management for experimental biology.</a> Nat Methods. 9, 245-253 (2012).</li><li>Culver, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+hypoxia-induced+NF-kappaB.">Mechanism of hypoxia-induced NF-kappaB.</a> Mol Cell Biol. 30, 4901-4921 (2010).</li><li>Darzynkiewicz, Z., Traganos, F., Zhao, H., Halicka, H. D., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytometry+of+DNA+replication+and+RNA+synthesis:+Historical+perspective+and+recent+advances+based+on+"click+chemistry.">Cytometry of DNA replication and RNA synthesis: Historical perspective and recent advances based on "click chemistry.</a> Cytometry A. 79, 328-337 (2011).</li></ol>

J.R.S. is Founder of Glencoe Software, Inc., an open-source US-based commercial company that contributes to OMERO.

We have described our use of an RNA imaging kit to examine the effects of hypoxia on RNA synthesis in osteosarcoma (U2OS) cells. This technique is relatively straightforward and provides data about global transcription at a single cell level. We modified the conventional protocol for this kit to incorporate labeling in a hypoxia chamber. To our knowledge this is the first report of the use of this technique to measure changes in RNA synthesis in hypoxia. The RNA imaging kit we used is suitable for experimentation in hypoxia since it requires no radioactive isotopes and is technically compatible with the limitations of working in a controlled atmosphere workstation. Common problems with this technique concern efficient fixation and labeling of the sample. It is extremely important that the PFA used to fix the sample is fresh and the wash steps that follow the &#39;click&#39; reaction are performed as described to ensure low background staining of the sample. If background fluorescence becomes a problem it is recommended to wash once more with 1 ml RNA imaging kit reaction rinse buffer after step 2.7.4.
The RNA imaging kit mentioned here represents a significant advance in RNA imaging technology in terms of ease of use and specificity and speed of reaction. This technique is primarily limited by the time it takes to label the sample. The RNA imaging kit requires a 1 hr labeling period that prevents the examination of rapid changes in global RNA that would usually occur within this timeframe. It is for this reason our first time point is restricted to 1 hr and 15 min. The technique is associated with few other limitations that largely relate to reagent compatibility with other fluorescent markers, for example, this RNA imaging kit is not compatible with phalloidin staining.
All existing approaches of studying RNA synthesis in cells are based on incorporation of modified nucleosides into the nascent RNA and the detection of incorporated labels by different means. The pioneering approach was based on using radioactively labeled RNA precursors followed by detection with autoradiography. This method led to a discovery of cell cycle stages and other important findings; however, it has a lot of disadvantages such as the cumbersome nature of radioactivity work, long exposure times and low resolution images of autoradiography6. The next advance in the field exploited means of immunochemistry to eliminate the necessity of radioactivity. RNA was labeled by incorporation of BrU followed by immunochemistry detection. However, efficient antibody binding required nucleic acid denaturation to improve access to DNA or RNA that caused loss of cell morphology and damaged the epitopes of many proteins, preventing their further detection with fluorescently labeled antibodies13. The introduction of &#39;click chemistry&#39; technology simplified the detection step and the procedure compared to autoradiography and immunochemistry. The technology is based on azide-alkyne Huisgen cycloaddition reaction where terminal alkyne of 5-ethyniluridine binds with azide conjugated with the fluorophore in presence of Cu(I). The reaction is specific, rapid, does not require any additional steps, and is easily compatible with immunochemical detection of other cell constituents.
Using this RNA imaging technology we showed that cells, in the same monolayer population, have different RNA synthesis levels in response to hypoxia. We restricted our experiment to examine the effect of RNA production only; however, it is wholly possible with this RNA imaging kit to perform modified, additional multiplex reactions that allow for a more complex analysis of RNA production. For example, secondary staining using an antibody specific for markers of active transcription such as levels of phosphorylated RNA polymerase II or even components of the translation machinery to give an indication of the amount of this newly produced RNA that is converted into protein are possible. Additional proteins, such as Actin, a protein that should not change significantly with hypoxia, could be tested as an additional control. Furthermore, different cell types could be tested, as it is very likely that different cells will have different RNA synthesis rates and even different responses to hypoxia.
Use of this RNA imaging kit is quite straightforward provided the steps are performed as described. Critical steps in the protocol involve cell plating, cell fixation and staining and image acquisition. It is important to plate the cells at the described density to prevent over or under confluence at experimentation which will significantly affect the result. It is also important to use fresh fixative, ensuring the best &#39;snapshot&#39; of the cell is taken and to take care when washing the cells after staining to reduce the background to a level that is acceptable for efficient analysis. Finally, the method of image acquisition is vitally important to achieve images that are of a good enough quality for subsequent analysis. We recommend using the best light microscope available to examine samples optically such as the microscope used in this protocol which is available at most research intensive centers globally.

Hypoxia (low oxygen tensions) occurs when the normal oxygen supply to a tissue is disturbed. Environmental oxygen is both a nutrient and a signaling molecule, providing important cues for many cell types. Changes in environmental oxygen are sensed by a group of dioxygenases that control the activity of an essential transcription factor family known as the Hypoxia Inducible Factors (HIFs). The HIFs are composed of two subunits, &#945; and &#946;. There are three known isoforms of HIF-&#945; (1, 2, and 3) and multiple splice variants of HIF-1&#946;. HIF-1&#946; is constitutively expressed and not regulated by environmental oxygen levels1. The HIF-&#945; family members are dynamically regulated by a class of prolyl-hydroxylases (PHDs) and the Factor Inhibiting HIF (FIH); both of which require oxygen as a co-factor to catalyze the hydroxylation of HIF-&#945;2,3. In normoxia the HIF-&#945; family members are hydroxylated and tagged for proteosomal degradation by the E3-Ligase, von Hippel Lindau (vHL). In hypoxia the PHDs and FIH are inactive or have a reduced activity. The HIF-&#945; isoforms become stabilized, form a heterodimer with HIF-1&#946;, and effect the transcription of genes that provide the cellular response to the hypoxic environment (Figure 1A)4.
Current techniques for RNA analysis focus on the quantification of averaged values across a given cell population. Cells respond to a hypoxic stimulus by initiating the transcription of a myriad of genes that allow them to adapt to their hostile environment5. However, hypoxia often exists as a gradient, and cells in a hypoxic environment are not subject to a uniform hypoxic stimulus. We describe an implementation of the Click-iT RNA imaging kits in an oxygen controlled workstation to examine global RNA synthesis at the single cell level in hypoxia.
The RNA imaging kit uses an alkyne-modified nucleoside, 5-ethynyl uridine (EU) and chemoselective ligation to enable detection of global RNA synthesis temporally and spatially in cells and tissues6.&#160; Briefly, cells are treated with hypoxia and cultured in the presence of EU. They are then fixed and permeabilized and EU incorporation into nascent RNA is detected by chemoselective ligation of EU with an azide containing dye. A typical workflow for this reaction is shown in Figure 1B. We used the RNA imaging kit to examine RNA synthesis at the single cell level that resulted from treatment with hypoxia.
The small size of the alkyne tag enables efficient incorporation of the modified nucleoside into RNA specifically. The chemoselective ligation or &#39;click&#39; reaction is highly efficient, fast and specific7-10. All of the reaction components are bioinert and the reaction requires no extreme temperatures or solvents. The click reaction negates the requirement for conventional radiolabelling and allows direct visualization of the results since the output is light. In addition, the detection molecule can easily penetrate complex samples allowing for multiplex analysis including antibodies for the detection of RNA-interactive proteins. This RNA imaging assay is compatible with organic dyes including Alexa Fluor and fluorescein (FITC).
We measured the change in RNA synthesis following treatment of our cells using an implementation of the open microscopy environment for remote objects (OMERO). OMERO is open-source software, available at <a href="http://openmicroscopy.org/">http://openmicroscopy.org/</a> . This microscope image visualization and analysis software enables access to, and use of a wide range of biological data, including the management of multidimensional, heterogeneous datasets. The client application allows remote visualization and analysis of complex biological image data11; we used it to quantify the visual changes in global and single cell RNA synthesis. These data and the steps required to analyze our RNA imaging experiment using this microscope image visualization and analysis software are shown below.
We looked at changes in global RNA synthesis following the treatment of human osteosarcoma (U2OS) cells with hypoxia for up to 24 hr. In all conditions, we detected cell-to-cell variation in the level of RNA production. Short times of hypoxia exposure did not result in significant changes to the level of nascent RNA in cells. However, exposure to 24 hr&#160;of hypoxia resulted in a significant increase in the amount of RNA produced in cells. Most of the cellular responses to hypoxia are measured following prolonged periods of exposure, such as 4 to 24 hr. However, some mechanisms are put in place much earlier, for example; NF-&#954;B activation occurs within 5-15 min of hypoxia exposure12. Investigating shorter hypoxia exposure times is therefore warranted and could detract from more complicated responses such as cell cycle and apoptosis.

<table border="0" cellpadding="0" cellspacing="0" style="width:1313px;" width="1314">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:236px;">U-2 OS Primary Cells</td>
			<td style="width:92px;">European Collection of Cell Cultures</td>
			<td align="right" style="width:115px;">92022711</td>
			<td style="width:168px;"></td>
			<td style="width:703px;"><a href="http://www.hpacultures.org.uk/products/celllines/generalcell/search.jsp?searchtext=u2os&amp;dosearch=true">http://www.hpacultures.org.uk/products/celllines/generalcell/search.jsp?searchtext=u2os&#38;dosearch=true</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Gibco</td>
			<td align="right">14190094</td>
			<td></td>
			<td><a href="http://products.invitrogen.com/ivgn/product/14190094">http://products.invitrogen.com/ivgn/product/14190094</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Gibco</td>
			<td align="right">41966029</td>
			<td></td>
			<td><a href="https://products.invitrogen.com/ivgn/product/41966029?ICID=search-41966029">https://products.invitrogen.com/ivgn/product/41966029?ICID=search-41966029</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Gibco</td>
			<td align="right">10270106</td>
			<td></td>
			<td><a href="http://products.invitrogen.com/ivgn/product/10270106?ICID=search-10270106">http://products.invitrogen.com/ivgn/product/10270106?ICID=search-10270106</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td>Lonza</td>
			<td>DE17-603E</td>
			<td></td>
			<td><a href="http://www.lonza.com/products-services/bio-research/cell-culture-products/reagents/antibiotics-antimycotics/penicillin-streptomycin.aspx">http://www.lonza.com/products-services/bio-research/cell-culture-products/reagents/antibiotics-antimycotics/penicillin-streptomycin.aspx</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17-605E</td>
			<td></td>
			<td><a href="http://www.lonza.com/products-services/bio-research/cell-culture-products/reagents/cell-culture-reagents/l-glutamine-200mm.aspx">http://www.lonza.com/products-services/bio-research/cell-culture-products/reagents/cell-culture-reagents/l-glutamine-200mm.aspx</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.05% Trypsin-EDTA (1%)</td>
			<td>Gibco</td>
			<td>25300062</td>
			<td></td>
			<td><a href="http://products.invitrogen.com/ivgn/product/25300062">http://products.invitrogen.com/ivgn/product/25300062</a></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Hypoxia Chamber</td>
			<td>Ruskinn</td>
			<td>InVivo2 300</td>
			<td></td>
			<td><a href="http://www.ruskinn.com/products/invivo2300">http://www.ruskinn.com/products/invivo2300</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Click-iT assay Kit</td>
			<td>Invitrogen</td>
			<td>C-10329/C10330</td>
			<td></td>
			<td>http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catDisplayStyle&#38;catKey=601107&#38;filterDispName=Nascent%2BRNA%2BDetection%2B%2526amp%253B%2BCapture%2BKits&#38;_bcs_=H4sIAAAAAAAAAMWP3WrDMAyFn8Y3MwtOUrrcZg0dpVAGZbs3jpYIEjvYSoLffvK6jbKV3Q6EhP44%0A37nPhaqevWtnQ0GKYivP4Bc0EP6Y90STKGtR7DnWdc3QLkjedWAz40YeBiTgMgdOYDn1boSsp3Hg%0Ad1GUKVRFfobUqwfFJVc5H1aFyu%2B4O%2BJFV48s9SjrEHT8pT19Av4EKOtK8RqXzn4BvJw56RY9GEqr%0A7wdR7s3YirI5vD6djKYGwzTouNMEnfOREXh4hMgXie2a%2F00P4aYBLsV2czFyms0AaGRtsJUNEOuj%0As9fWrB5iwCCT5X92%2BEF9y%2BI7x9UoYCgCAAA%3D</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pFA</td>
			<td>Sigma</td>
			<td>76240</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/fluka/76240?lang=en&amp;region=GB">http://www.sigmaaldrich.com/catalog/product/fluka/76240?lang=en&#38;region=GB</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma</td>
			<td>X-100</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/x100?lang=en&amp;region=GB">http://www.sigmaaldrich.com/catalog/product/sial/x100?lang=en&#38;region=GB</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10M NaOH</td>
			<td>Sigma</td>
			<td>72068</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/72068?lang=en&amp;region=GB">http://www.sigmaaldrich.com/catalog/product/sigma/72068?lang=en&#38;region=GB</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">37% HCl</td>
			<td>VWR</td>
			<td>20252.335</td>
			<td></td>
			<td><a href="https://uk.vwr.com/app/search/Search?keywords=%2720252.244%27%20%2720252.290%27%20%2720252.324%27%20%2720252.335%27%20%2720252.368%27%20%2720252.420%27%20%2720252.448%27%20%2720252.980%27&amp;searchOp=or">https://uk.vwr.com/app/search/Search?keywords=%2720252.244%27%20%2720252.290%27%20%2720252.324%27%20%2720252.335%27%20%2720252.368%27%20%2720252.420%27%20%2720252.448%27%20%2720252.980%27&#38;searchOp=or</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VectaShield Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1400</td>
			<td></td>
			<td><a href="http://www.vectorlabs.com/catalog.aspx?prodID=1483">http://www.vectorlabs.com/catalog.aspx?prodID=1483</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Actinomycin D</td>
			<td>Sigma</td>
			<td>A9415</td>
			<td></td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a9415?lang=en&amp;region=GB">http://www.sigmaaldrich.com/catalog/product/sigma/a9415?lang=en&#38;region=GB</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Deltavision Core Microscope</td>
			<td>Applied Precision</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.appliedprecision.com/">http://www.appliedprecision.com/</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">softWoRx [Refered to in text as image processing software]</td>
			<td>Applied Precision</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.api.com/softworx.asp">http://www.api.com/softworx.asp</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CoolSnap HQ2 CCD Camera</td>
			<td>Photometrics</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.photometrics.com/products/ccdcams/coolsnap_hq2.php">http://www.photometrics.com/products/ccdcams/coolsnap_hq2.php</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Open Microscopy Environment for Remote Objects (OMERO) [refered to in text as microscope image visualisation and analysis software]</td>
			<td>OME</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://openmicroscopy.org/">http://openmicroscopy.org/</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SigmaPlot v12.0 [Refered to in text as data graphing software]</td>
			<td>Systat Software Inc.</td>
			<td>N/A</td>
			<td></td>
			<td><a href="http://www.sigmaplot.co.uk/products/sigmaplot/sigmaplot-details.php">http://www.sigmaplot.co.uk/products/sigmaplot/sigmaplot-details.php</a></td>
		</tr>
	</tbody>
</table>

analysis of global rna synthesis at the single cell level following hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

A schematic of the RNA imaging kit reaction is shown in Figure 1B. We used this reaction to quantitatively determine total RNA synthesis in U2OS cells following treatment with hypoxia on both an individual (single cell) and global level (population of cells). The cells were treated with hypoxia and cultured in the presence of EU. They were then fixed and permeabilized and EU incorporation was detected by chemoselective ligation of EU with an azide containing dye. This &#39;click&#39; reaction results in a fluorescence emission that can be detected and quantified using fluorescence light microscopy. Figure 2A shows a typical result from this experiment. Fluorescently labeled cells are pseudocolored red and the image has been opened in the open microscopy environment for remote objects (OMERO). This microscope image visualization and analysis software has a built-in ROI tool that can be used to select individual sub-cellular structures and quantitatively analyze image intensity within the chosen region. A typical example of the data output following the use of this tool in the client program can be seen in Figure 2B; individual cell intensities are shown alongside a representative image from the experiment that serves as a visual check to ensure data validity.
The individual cell fluorescence intensities can be plotted as a scatter chart to show the distribution of the individual cellular intensities within the treated cell population. An example of this type of graph is seen in Figure 2C. Plotting intensity data in this way is useful because it allows a rapid comparison of treatment results and clearly demonstrates the significant heterogeneity within a treatment cell population that is otherwise obscured using standard data representation. The response heterogeneity is interesting in this experiment because although a uniform hypoxic stimulus was applied to the cells, it is evident that every cell from the same monolayer population responds differently to this stimulus.
Figure 2D depicts the overall result from our experiment. Fluorescence intensities from all cells within a treatment population were averaged and then plotted in this bar chart. A student&#39;s t-test has been used to calculate the statistical probability of each treatment group differing significantly from the control. Interestingly, these data show that there is a statistically significant increase in global RNA synthesis over time when U2OS cells are treated with hypoxia (at times greater than 1 hr 30 min. *p &#60; 0.01; **p &#60; 0.05 and ***p &#60; 0.001 and n &#62; 30). These data show that following longer periods of hypoxia treatment, the cell focusses its efforts on the production of RNA even in this hostile hypoxic environment. Actinomycin D treatment completely ablates the fluorescence intensity as expected.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51420/51420fig1highres.jpg" src="/files/ftp_upload/51420/51420fig1.jpg" /> 
	Figure 1. A. The HIF system responds to changes in cellular oxygen. In this simplified schematic hypoxia stabilizes the HIF heterodimer by preventing HIF-&#945; hydroxylation (-OH) by the prolyl hydroxylase enzymes (PHDs) and its subsequent polyubquitination by von-Hippel Lindau protein (vHL). HIF stabilization results in the activation of myriad genes that help the cell respond to cellular hypoxia. B. RNA synthesis can be detected using the Click-IT assay; cells are labelled with EU after treatment the Click reaction detects RNA synthesis which can be quantified by fluorescence intensity and light microscopy. <a href="https://www.jove.com/files/ftp_upload/51420/51420fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51420/51420fig2highres.jpg" src="/files/ftp_upload/51420/51420fig2.jpg" /> 
	Figure 2. A. Screenshot from OMERO to show typical microscopic output following RNA labelling. This microscope image visualization and analysis software can quickly extract intensity data on an individual cell level using its inbuilt ROI macro. A screenshot of the output from this command is shown in B. Global RNA synthesis can be inferred from individual cell intensity, the range of which can be seen in scatter plot C. A formal representation of these aggregate data can be seen D. *p &#60; 0.01; **p &#60; 0.05 and ***p &#60; 0.001 and n &#62; 30. <a href="https://www.jove.com/files/ftp_upload/51420/51420fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cell Culture Reagents</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) pH 7.2 - 7.6</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagles Medium (DMEM)</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS) filter sterilized</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA (1%)</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
		</tr>
		<tr>
			<td>Actinomycin D (1 &#956;g/&#956;l stock)</td>
		</tr>
		<tr>
			<td>Cell Culture Equipment</td>
		</tr>
		<tr>
			<td>Sterile Pasteur Pipettes</td>
		</tr>
		<tr>
			<td>A range of cell culture-treated plastic ware suitable for the maintenance and treatment of cells in culture</td>
		</tr>
		<tr>
			<td>Water bath set at 37 &#176;C</td>
		</tr>
		<tr>
			<td>Laminar air flow hood</td>
		</tr>
		<tr>
			<td>Incubator set at 37 &#176;C, 5% CO2</td>
		</tr>
		<tr>
			<td>Hypoxia Workstation set at 37 &#176;C, 5% CO2, 1% O2</td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
		</tr>
		<tr>
			<td>precision forceps</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
		</tr>
	</tbody>
</table>
Table 1. Reagents and equipment required for culturing of U2OS cells.
<table border="0" cellpadding="0" cellspacing="0" width="514">
	<tbody>
		<tr>
			<td rowspan="2">Reaction Components</td>
			<td colspan="6">Number of Coverslips</td>
		</tr>
		<tr>
			<td>1</td>
			<td>2</td>
			<td>4</td>
			<td>5</td>
			<td>6</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Click-iT RNA reaction buffer (Component C)</td>
			<td>428 &#956;l</td>
			<td>856 &#956;l</td>
			<td>1.7 ml</td>
			<td>2.1 ml</td>
			<td>2.58 ml</td>
			<td>4.3 ml</td>
		</tr>
		<tr>
			<td>CuSO4 (Component D)</td>
			<td>20 &#956;l</td>
			<td>40 &#956;l</td>
			<td>80 &#956;l</td>
			<td>100 &#956;l</td>
			<td>120 &#956;l</td>
			<td>200 &#956;l</td>
		</tr>
		<tr>
			<td>Alexa Fluor Azide (Prepared in Step 2.3)</td>
			<td>1.8 &#956;l</td>
			<td>3.7 &#956;l</td>
			<td>7.4 &#956;l</td>
			<td>9.3 &#956;l</td>
			<td>11.28 &#956;l</td>
			<td>18.8 &#956;l</td>
		</tr>
		<tr>
			<td>Click-iT reaction buffer additive (Prepared in step 2.3)</td>
			<td>50 &#956;l</td>
			<td>100 &#956;l</td>
			<td>200 &#956;l</td>
			<td>250 &#956;l</td>
			<td>300 &#956;l</td>
			<td>500 &#956;l</td>
		</tr>
		<tr>
			<td>Approximate Total Volume</td>
			<td>500 &#956;l</td>
			<td>1 ml</td>
			<td>2 ml</td>
			<td>2.5 ml</td>
			<td>1.5 ml</td>
			<td>5 ml</td>
		</tr>
	</tbody>
</table>
Table 2. RNA imaging kit reaction cocktails: Reference table detailing ratio of master mix components for RNA imaging kit reaction according to the number of coverslips to be stained.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Fluorophore</td>
			<td>Excitation (nm)</td>
			<td>Emission (nm)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>495</td>
			<td>519</td>
		</tr>
		<tr>
			<td>Hoechst 33342, bound to DNA</td>
			<td>350</td>
			<td>461</td>
		</tr>
	</tbody>
</table>
Table 3. Fluorescence/excitation emission maxima: Approximate fluorescence/excitation emission maximal for Alexa Fluor dye and Hoechst 33342 bound to DNA.

Watch this Scientific Journal Video about Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia at JoVE.com

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

analysis-global-rna-synthesis-at-single-cell-level-following

Research

JoVE Journal

Biology

12.2K Views.  University of Dundee, UK. We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Video: Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &#955; DNA at the Single Molecule Level

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. In single molecule assays for studying DNA-protein interactions, there are two important factors for consideration: the DNA substrate with enough length for easy observation and labeling a protein with a suitable fluorescent probe. 48.5 kb &#955; DNA is a good candidate for the DNA substrate. Quantum dots (Qdots), as a class of fluorescent probes, allow long-time observation (minutes to hours) and high-quality image acquisition. In this paper, we present a protocol to study DNA-protein interactions at the single-molecule level, which includes preparing a site-specifically modified &#955; DNA and labeling a target protein with streptavidin-coated Qdots. For a proof of concept, we choose ORC (origin recognition complex) in budding yeast as a protein of interest and visualize its interaction with an ARS (autonomously replicating sequence) using TIRFM. Compared with other fluorescent probes, Qdots have obvious advantages in single molecule studies due to its high stability against photobleaching, but it should be noted that this property limits its application in quantitative assays.

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &amp;#955; DNA at the Single Molecule Level

Here, we present a protocol to study DNA-protein interactions by total internal reflection fluorescence microscopy (TIRFM) using a site-specifically modified &#955; DNA substrate and a Quantum-dot labeled protein.

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. ...